Mechanism Of The KIKER Cluster In The Inactivation Of HERG Channels

hERG（human ether-a-go-go-related gene）channel plays a key role in regulating cardiac excitability and maintaining normal heart rhythm.Studying the gating mechanism of hERG channel is of great significance to understand the structural basis of its unique function and the molecular basis of the pathogenesis of abnormal channel function.In the previous study of our group,it was found that there is still inactivation in the hERG channel after the C-type inactivation has been removed.It is speculated that there may be another inactivation of the hERG channel-N-type inactivation.The“ball-chain”model often explains N-type inactivation,the key sphere exists at the N-terminus.When the channel is activated,the sphere is dragged into the pore by the chain and binds to the receptor in the channel to block the channel and cause inactivation.The KIKER cluster of five consecutive charged residues at the N-terminus is a key region in the N-terminal proximal domain of the hERG channel.The study speculates that the KIKER cluster is a sphere with a ball-chain structure,and the receptor may be composed of three regions on the pore domain of the hERG channel:Provision of sites on top and middle of water cavity,intracellular gate/bundle crossing gate.The study investigated the gating mechanism of the hERG channels by analyzing the channel structure by disulfide bridge method and Pymol.The main findings of the study are as follows:1.Firstly,cysteine mutations were introduced into the proximal domain E365 of the KIKER cluster,T623,S624 at the top of the water cavity,Y652,F656 in the middle of the water cavity,and Q664 of the intracellular gate/bundle crossing gate,to generate six single-cysteine hERG mutants channels,and the resulting disulfide bonds were opened by applying of reducing agent DTT.The results showed that there was no significant difference between the tail current increase of DTT irrigated by the six single-mutated channels and that of the wild-type hERG channel,the single-mutated channel did not produce disulfide bonds that were different from those of the wild-type hERG channel.However,the channel tail current after DTT irrigation was significantly different from that of the single mutation channel without DTT irrigation,which confirmed that the reducing agent DTT did indeed destroy disulfide bonds.2.Then,E365 of the KIKER cluster was connected with T623,S624 at the top of the water cavity,Y652,F656 in the middle of the water cavity,and Q664 of the intracellular gate/bundle crossing gate to form five double mutation channels E365C-T623C,E365C-S624C,S624C-Y652C,E365C-F656C,E365C-Q664C were perfused with DTT,and the results showed that the E365C-Q664C double mutant channel had the largest increase in tail current after perfusion with DTT.To verify the accuracy of the experiment,the E365C-T623C and E365C-Q664C double mutant channels were perfused with DTT followed by the oxidant TBHO₂（which acts to create an oxidative environment for disulfide bond formation）.The results of double mutation showed that E365C-Q664C cells perfused with TBHO₂ can reduce the wake of the channel compared with the channel after perfusion with DTT,but the effect is not significant compared with the wild-type channel.3.The time course program was applied to the E365C-Q664C channel to prolong the activation time,the purpose is to create more opportunities to generate disulfide bonds and reduce the channel current,and apply TBHO₂ oxidant agent to further reduce the current,and finally,perfusion of the reducing agent DTT will increase the channel current more obviously.The results show that there is a highly significant difference between the addition of DTT to the channel under this voltage procedure and the infusion of TBHO₂,with a further reduction in current compared to the same channel under the steady-state activation protocol.4.Applying a rate-of-activation voltage program to the single-and double-mutant channels,the normalized value of the tail current of the E365C-Q664C channel was greater than that of the E365C-T623C and wild-type hERG channels.The large inactivation rate of the E365C-Q664C channel proves that the disulfide bond generated between these two sites is likely to be generated during the inactivation of the gating movement.In summary,the experimental results show that the E365C-Q664C channel forms a disulfide bond.We speculate that the gating inactivation movement of the hERG channel is:that the KIKER cluster at the N-terminus where E365 is located acts as the"ball-chain"in the N-type inactivation.N-type inactivation occurs when the sphere moves to and interacts with the intracellular gate/bundle crossing gate on the S6 helix when the channel transitions to the deactivated state.